The C/EBP family of transcription factors (C/EBP-alpha, C/EBP-beta, C/EBP-delta and CHOP-10) plays an important role in adipocyte differentiation. These factors appear to function sequentially in a cascade that leads to expression of C/EBP-alpha, a pleiotropic transcriptional activator of numerous genes that produce the adipocyte phenotype. The long-term goal of this research is to understand the role of C/EBP-beta in this cascade. By elucidating these events it should be possible to identify sites at which adipocyte development and thereby, obesity and its consequences, can be controlled. When induced to differentiate, growth-arrested 3T3-L1 preadipocytes synchronously re-enter the cell cycle, undergo several rounds of "mitotic clonal expansion" and then express adipocyte genes and acquire adipocyte characteristics. Mitotic clonal expansion is required for subsequent differentiation for reasons that have yet to be defined. C/EBP-beta, a transcriptional activator of the C/EBP-alpha gene, is expressed immediately upon induction of differentiation, but does not acquire DNA binding activity until much later as the preadipocytes pass the G1-S check-point of the cell cycle. Concomitantly, C/EBP-beta becomes phosphorylated, loses its inhibitory association with CHOP-10 (a dominant-negative C/EBP) and translocates to centromeres. I propose to determine the role of C/EBP-beta in the mitotic clonal expansion process of the adipocyte differentiation program. The SPECIFIC AIMS are to: -identify the phosphorylation site(s) in C/EBP-beta that produce DNA binding activity as preadipocytes traverse the G1-S check-point during mitotic clonal expansion. -identify the kinase(s) that catalyzes this/these phosphorylation/s and to determine how the kinase(s) is regulated. -determine the role of C/EBP-beta in mitotic clonal expansion and how phosphorylation of C/EBP-beta affects this role.